1. Field of the Invention
The invention relates to a method used in fluorescence microscopy, especially laser scanning microscopy, in general, and to the use of dyes for image evaluation, in particular.
2. Description of Related Art
A conventional application of light microscopy for investigation of biological preparations is fluorescence microscopy which is discussed in the “Handbook of Biological Confocal Microscopy, Second Edition”, Pawley, Plenum Press, 1995. Specific dyes are used in fluorescence microscopy for specific marking of tissues, cells, cell parts, or other materials.
The emitted photons of a specific energy excite the dye molecules from the ground state to an excited state by absorption of a photon. This excitation is generally referred to as one-photon absorption (See FIG. 1a). The dye molecules so excited can return in different ways to the ground state. In fluorescence microscopy the transition with emission of a fluorescence photon is most important. The wavelength of the emitted photon is generally red-shifted based on the Stokes Shift in comparison to the excitation radiation and therefore has a greater wavelength. The Stokes Shift permits separation of the fluorescence radiation from the excitation radiation.
A multiphoton excitation is shown in FIG. 1b. The fluorescence light is split and observed separately from the excitation radiation with appropriate dichroic beam splitters in combination with block filters. The depiction of individual cell parts stained with different dyes is possible on this account. However, several parts of a preparation, in principle, can also be simultaneously stained with different dyes by specifically adding dyes resulting in multiple fluorescence. Special dichroic beam splitters are again used to distinguish the fluorescence signals emitted by the individual dyes.
The prior art is explained as follows based on the example of a confocal laser scanning microscope (LSM) such as that show schematically in FIG. 2. An LSM is divided into essentially four modules: light source L, scan module S, detection unit D and microscope M. These modules are further described below. U.S. Pat. No. 6,167,173 also provides a detailed explanation of the LSM and is incorporated by reference herein as if reproduced in its entirety.
For specific excitation of different dyes in one preparation, different wavelengths are used in an LSM laser. The choice of excitation wavelength is guided according to the absorption properties of the dyes being investigated. The excitation radiation is generated in the light source module L. Different lasers 13.1 and 13.2 are used here (for example, argon, argon-krypton, TiSa lasers). Selection of the wavelength and adjustment of the intensity of the required excitation wavelength additionally occurs in the light source module L, for example by using an acousto-optic crystal 15.1 and 15.2. The laser radiation then goes to the scan module S via a fiber 14.1 and 14.2 and/or appropriate mirror arrangement.
The laser radiation generated in the light source is focused into the preparation with limited diffraction with objective 4 via the scanner 23, scan optics 16 and 17 and tube lens 9. The focus scans the sample in the x-y direction point-like. The pixel residence times during scanning over the sample are generally in the range of less than a microsecond to a few seconds.
During confocal detection (descanned detection) of the fluorescence light, the light emitted from the focal plane (specimen) 5 and from the overlying and underlying planes goes to a dichroic beam splitter (MDB) 24 via the scanner 23. This separates the fluorescence light from the excitation light. The fluorescence light is then focused on a diaphragm (confocal diaphragm/pinhole) 29, which is situated precisely in a plane conjugated to the focal plane. Fluorescence light fractions outside of the focus are suppressed on this account. By varying the diaphragm size the optical resolution of the microscope can be adjusted. Behind the diaphragm there is an additional dichroic block filter (EF) 30 that again suppresses the excitation radiation. After passing through the block filter, the fluorescence light is measured by means of a point detector (PMT) 31.
During use of multiphoton absorption, excitation of dye fluorescence occurs in a small volume on which the excitation intensity is particularly high. This region is only slightly larger than the detected region during use of a confocal arrangement. The use of a confocal diaphragm can therefore be omitted and detection can occur directly after the objective 5 in the nondescanned detection region 71 of the microscope.
In another arrangement for detection of dye fluorescence excited by multiphoton absorption, descanned detection again occurs, but this time the pupil of the objective is imaged in the detection unit (nonconfocal descanned detection) D.
Only the plane (optical section) that is situated in the focal plane of the objective is detected by a three-dimensionally illuminated image by both detection arrangements in conjunction with the corresponding one photon or multiphoton absorption. By marking several optical sections in the x-y plane at different depths z of the sample, a three-dimensional image of the sample can then be generated in computer-controlled fashion. The LMS is therefore suitable for investigating thick preparations. The excitation wavelengths are determined by the employed dye with specific absorption properties. Dichroic filters 30 adjusted to the emission properties of the dye ensure that only the fluorescence light emitted by the corresponding dye is measured by the point detector.
In biomedical applications several different cells or cell regions are now marked with different dyes simultaneously (multifluorescence). The individual dyes can be detected separately with the prior art based either on different absorption properties or emission properties (spectra). For this purpose additional splitting of the fluorescence light from several dyes occurs with the secondary beam splitters (DBS) 28 and a separate detection of the individual dye emissions in separate point detectors (PMT x) 31. Flexible adjustment of detection and excitation to corresponding new properties by the user is not possible with the arrangement described above. New dichroic beam splitters and block filters must instead be created for each (new) dye.
If the emission spectra of two dyes overlap, the previous detection devices reach their limits. In order to avoid overlap between two dyes, the spectral detection range must be restricted. The range in which the two dyes overlap is simply cut out for this purpose and not detected. The efficiency of the detection unit therefore deteriorates. An equal signal-to-noise ratio can only be achieved by increasing the excitation power, through which preparation damage can occur. Nowadays a maximum of up to six different dye probes are therefore simultaneously used, since the dyes otherwise could not be separated owing to the strongly overlapping emission bands.
Previously dyes have been modified so that they either differ from each in their absorption properties or in their emission properties. FIG. 3 shows the emission spectra of different typical dyes. The emission signal is plotted as a function of wavelength. The dyes denoted 1 to 4 differ in position and form of their emission spectra. These dyes, however, are in most cases toxic for living preparations. Investigation of the evolution of cell structure in living preparations is therefore impossible. In the late 90s, dyes occurring in nature, the so-called fluorescing proteins (GFP, YFP, CFP, TOPAS, GFT, RFP) were discovered by Clonetech, Mountain View, Calif. www.clontech.com. Fluorescence dyes for specific marking of preparations are used in all of the aforementioned systems.